17beta-Hydroxysteroid dehydrogenase type 11 (17beta-HSD11) is a member of the short-chain dehydrogenase/reductase family involved in the activation and inactivation of sex steroid hormones. We recently identified 17beta-HSD11 as a gene that is efficiently regulated by peroxisome proliferator-activated receptor-alpha PPARalpha in the intestine and the liver [Motojima K (2004) Eur J Biochem271, 4141-4146]. In this study, we characterized 17beta-HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver. For this purpose, specific antibodies against 17beta-HSD11 were obtained. Western blotting analysis showed that administration of a peroxisome proliferator-activated receptor-alpha agonist induced 17beta-HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice. A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein-tagged 17beta-HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced. A pulse-chase experiment suggested that 17beta-HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum. Immunohistochemical analysis using tissue sections showed that 17beta-HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures. A subcellular fractionation study of liver homogenates confirmed that 17beta-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator-activated receptor-alpha agonist. Taken together, these data indicate that 17beta-HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17beta-HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments.